Ongoing Cooperative Engagement Facilitates Agile Pandemic and Outbreak Response: Lessons Learned Through Cooperative Engagement Between Uganda and the United States.

Pathogens threaten human lives and disrupt economies around the world. This has been clearly illustrated by the current COVID-19 pandemic and outbreaks in livestock and food crops. To manage pathogen emergence and spread, cooperative engagement programs develop and strengthen biosafety, biosecurity, and biosurveillance capabilities among local researchers to detect pathogens. In this case study, we describe the efforts of a collaboration between the Los Alamos National Laboratory and the Uganda Virus Research Institute, the primary viral diagnostic laboratory in Uganda, to implement and ensure the sustainability of sequencing for biosurveillance. We describe the process of establishing this capability along with the lessons learned from both sides of the partnership to inform future cooperative engagement efforts in low- and middle-income countries. We found that by strengthening sequencing capabilities at the Uganda Virus Research Institute before the COVID-19 pandemic, the institute was able to successfully sequence SARS-CoV-2 samples and provide data to the scientific community. We highlight the need to strengthen and sustain capabilities through in-country training, collaborative research projects, and trust.

The Age of Next-Generation Therapeutic-Microbe Discovery: Exploiting Microbe-Microbe and Host-Microbe Interactions for Disease Prevention.

Humans are considered "superorganisms," harboring a diverse microbial collective that outnumbers human cells 10 to 1. Complex and gravely understudied host- and microbe-microbe interactions-the product of millions of years of host-microbe coevolution-govern the superorganism in almost every aspect of life functions and overall well-being. Abruptly disrupting these interactions via extrinsic factors has undesirable consequences for the host. On the other hand, supplementing commensal or beneficial microbes may mitigate perturbed interactions or enhance the interactive relationships that ultimately benefit all parties. Hence, immense efforts have focused on dissecting the innumerable host- and microbe-microbe relationships to characterize if a "positive" or "negative" interaction is at play and to exploit such behavior for broader implications. For example, microbiome research has worked to identify and isolate naturally antipathogenic microbes that may offer therapeutic potential either in a direct, one-on-one application or by leveraging its unique metabolic properties. However, the discovery and isolation of such desired therapeutic microbes from complex microbiota have proven challenging. Currently, there is no conventional technique to universally and functionally screen for these microbes. With this said, we first describe in this review the historical (probiotics) and current (fecal microbiota or defined consortia) perspectives on therapeutic microbes, present the discoveries of therapeutic microbes through exploiting microbe-microbe and host-microbe interactions, and detail our team's efforts in discovering therapeutic microbes via our novel microbiome screening platform. We conclude this minireview by briefly discussing challenges and possible solutions with therapeutic microbes' applications and paths ahead for discovery.

Discovery of an Antarctic Ascidian-Associated Uncultivated Verrucomicrobia with Antimelanoma Palmerolide Biosynthetic Potential.

The Antarctic marine ecosystem harbors a wealth of biological and chemical innovation that has risen in concert over millennia since the isolation of the continent and formation of the Antarctic circumpolar current. Scientific inquiry into the novelty of marine natural products produced by Antarctic benthic invertebrates led to the discovery of a bioactive macrolide, palmerolide A, that has specific activity against melanoma and holds considerable promise as an anticancer therapeutic. While this compound was isolated from the Antarctic ascidian Synoicum adareanum, its biosynthesis has since been hypothesized to be microbially mediated, given structural similarities to microbially produced hybrid nonribosomal peptide-polyketide macrolides. Here, we describe a metagenome-enabled investigation aimed at identifying the biosynthetic gene cluster (BGC) and palmerolide A-producing organism. A 74-kbp candidate BGC encoding the multimodular enzymatic machinery (hybrid type I-trans-AT polyketide synthase-nonribosomal peptide synthetase and tailoring functional domains) was identified and found to harbor key features predicted as necessary for palmerolide A biosynthesis. Surveys of ascidian microbiome samples targeting the candidate BGC revealed a high correlation between palmerolide gene targets and a single 16S rRNA gene variant (R = 0.83 to 0.99). Through repeated rounds of metagenome sequencing followed by binning contigs into metagenome-assembled genomes, we were able to retrieve a nearly complete genome (10 contigs) of the BGC-producing organism, a novel verrucomicrobium within the Opitutaceae family that we propose here as "Candidatus Synoicihabitans palmerolidicus." The refined genome assembly harbors five highly similar BGC copies, along with structural and functional features that shed light on the host-associated nature of this unique bacterium. IMPORTANCE Palmerolide A has potential as a chemotherapeutic agent to target melanoma. We interrogated the microbiome of the Antarctic ascidian, Synoicum adareanum, using a cultivation-independent high-throughput sequencing and bioinformatic strategy. The metagenome-encoded biosynthetic machinery predicted to produce palmerolide A was found to be associated with the genome of a member of the S. adareanum core microbiome. Phylogenomic analysis suggests the organism represents a new deeply branching genus, "Candidatus Synoicihabitans palmerolidicus," in the Opitutaceae family of the Verrucomicrobia phylum. The Ca. Synoicihabitans palmerolidicus 4.29-Mb genome encodes a repertoire of carbohydrate-utilizing and transport pathways, a chemotaxis system, flagellar biosynthetic capacity, and other regulatory elements enabling its ascidian-associated lifestyle. The palmerolide producer's genome also contains five distinct copies of the large palmerolide biosynthetic gene cluster that may provide structural complexity of palmerolide variants.

Parabacteroides distasonis: intriguing aerotolerant gut anaerobe with emerging antimicrobial resistance and pathogenic and probiotic roles in human health.

Parabacteroides distasonis is the type strain for the genus Parabacteroides, a group of gram-negative anaerobic bacteria that commonly colonize the gastrointestinal tract of numerous species. First isolated in the 1930s from a clinical specimen as Bacteroides distasonis, the strain was re-classified to form the new genus Parabacteroides in 2006. Currently, the genus consists of 15 species, 10 of which are listed as 'validly named' (P. acidifaciens, P. chartae, P. chinchillae, P. chongii, P. distasonis, P. faecis, P. goldsteinii, P. gordonii, P. johnsonii, and P. merdae) and 5 'not validly named' (P. bouchesdurhonensis, P. massiliensis, P. pacaensis, P. provencensis, and P. timonensis) by the List of Prokaryotic names with Standing in Nomenclature. The Parabacteroides genus has been associated with reports of both beneficial and pathogenic effects in human health. Herein, we review the literature on the history, ecology, diseases, antimicrobial resistance, and genetics of this bacterium, illustrating the effects of P. distasonis on human and animal health.

Democratization of fungal highway columns as a tool to investigate bacteria associated with soil fungi.

Bacteria-fungi interactions (BFIs) are essential in ecosystem functioning. These interactions are modulated not only by local nutritional conditions but also by the physicochemical constraints and 3D structure of the environmental niche. In soils, the unsaturated and complex nature of the substrate restricts the dispersal and activity of bacteria. Under unsaturated conditions, some bacteria engage with filamentous fungi in an interaction (fungal highways) in which they use fungal hyphae to disperse. Based on a previous experimental device to enrich pairs of organisms engaging in this interaction in soils, we present here the design and validation of a modified version of this sampling system constructed using additive printing. The 3D printed devices were tested using a novel application in which a target fungus, the common coprophilous fungus Coprinopsis cinerea, was used as bait to recruit and identify bacterial partners using its mycelium for dispersal. Bacteria of the genera Pseudomonas, Sphingobacterium and Stenotrophomonas were highly enriched in association with C. cinerea. Developing and producing these new easy-to-use tools to investigate how bacteria overcome dispersal limitations in cooperation with fungi is important to unravel the mechanisms by which BFIs affect processes at an ecosystem scale in soils and other unsaturated environments.

Bioinformatic and Mechanistic Analysis of the Palmerolide PKS-NRPS Biosynthetic Pathway From the Microbiome of an Antarctic Ascidian.

Complex interactions exist between microbiomes and their hosts. Increasingly, defensive metabolites that have been attributed to host biosynthetic capability are now being recognized as products of host-associated microbes. These unique metabolites often have bioactivity targets in human disease and can be purposed as pharmaceuticals. Polyketides are a complex family of natural products that often serve as defensive metabolites for competitive or pro-survival purposes for the producing organism, while demonstrating bioactivity in human diseases as cholesterol lowering agents, anti-infectives, and anti-tumor agents. Marine invertebrates and microbes are a rich source of polyketides. Palmerolide A, a polyketide isolated from the Antarctic ascidian Synoicum adareanum, is a vacuolar-ATPase inhibitor with potent bioactivity against melanoma cell lines. The biosynthetic gene clusters (BGCs) responsible for production of secondary metabolites are encoded in the genomes of the producers as discrete genomic elements. A candidate palmerolide BGC was identified from a S. adareanum microbiome-metagenome based on a high degree of congruence with a chemical structure-based retrobiosynthetic prediction. Protein family homology analysis, conserved domain searches, active site and motif identification were used to identify and propose the function of the ∼75 kbp trans-acyltransferase (AT) polyketide synthase-non-ribosomal synthase (PKS-NRPS) domains responsible for the stepwise synthesis of palmerolide A. Though PKS systems often act in a predictable co-linear sequence, this BGC includes multiple trans-acting enzymatic domains, a non-canonical condensation termination domain, a bacterial luciferase-like monooxygenase (LLM), and is found in multiple copies within the metagenome-assembled genome (MAG). Detailed inspection of the five highly similar pal BGC copies suggests the potential for biosynthesis of other members of the palmerolide chemical family. This is the first delineation of a biosynthetic gene cluster from an Antarctic microbial species, recently proposed as Candidatus Synoicihabitans palmerolidicus. These findings have relevance for fundamental knowledge of PKS combinatorial biosynthesis and could enhance drug development efforts of palmerolide A through heterologous gene expression.

Uncovering the Core Microbiome and Distribution of Palmerolide in Synoicum adareanum Across the Anvers Island Archipelago, Antarctica.

Polar marine ecosystems hold the potential for bioactive compound biodiscovery, based on their untapped macro- and microorganism diversity. Characterization of polar benthic marine invertebrate-associated microbiomes is limited to few studies. This study was motivated by our interest in better understanding the microbiome structure and composition of the ascidian, Synoicum adareanum, in which palmerolide A (PalA), a bioactive macrolide with specificity against melanoma, was isolated. PalA bears structural resemblance to a hybrid nonribosomal peptide-polyketide that has similarities to microbially-produced macrolides. We conducted a spatial survey to assess both PalA levels and microbiome composition in S. adareanum in a region of the Antarctic Peninsula near Anvers Island (64° 46'S, 64° 03'W). PalA was ubiquitous and abundant across a collection of 21 ascidians (3 subsamples each) sampled from seven sites across the Anvers Island Archipelago. The microbiome composition (V3-V4 16S rRNA gene sequence variants) of these 63 samples revealed a core suite of 21 bacterial amplicon sequence variants (ASVs)-20 of which were distinct from regional bacterioplankton. ASV co-occurrence analysis across all 63 samples yielded subgroups of taxa that may be interacting biologically (interacting subsystems) and, although the levels of PalA detected were not found to correlate with specific sequence variants, the core members appeared to occur in a preferred optimum and tolerance range of PalA levels. These results, together with an analysis of the biosynthetic potential of related microbiome taxa, describe a conserved, high-latitude core microbiome with unique composition and substantial promise for natural product biosynthesis that likely influences the ecology of the holobiont.

Salivary microbiomes of indigenous Tsimane mothers and infants are distinct despite frequent premastication.

BACKGROUND: Premastication, the transfer of pre-chewed food, is a common infant and young child feeding practice among the Tsimane, forager-horticulturalists living in the Bolivian Amazon. Research conducted primarily with Western populations has shown that infants harbor distinct oral microbiota from their mothers. Premastication, which is less common in these populations, may influence the colonization and maturation of infant oral microbiota, including via transmission of oral pathogens. We collected premasticated food and saliva samples from Tsimane mothers and infants (9-24 months of age) to test for evidence of bacterial transmission in premasticated foods and overlap in maternal and infant salivary microbiota. We extracted bacterial DNA from two premasticated food samples and 12 matched salivary samples from maternal-infant pairs. DNA sequencing was performed with MiSeq (Illumina). We evaluated maternal and infant microbial composition in terms of relative abundance of specific taxa, alpha and beta diversity, and dissimilarity distances. RESULTS: The bacteria in saliva and premasticated food were mapped to 19 phyla and 400 genera and were dominated by Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes. The oral microbial communities of Tsimane mothers and infants who frequently share premasticated food were well-separated in a non-metric multi-dimensional scaling ordination (NMDS) plot. Infant microbiotas clustered together, with weighted Unifrac distances significantly differing between mothers and infants. Infant saliva contained more Firmicutes (p < 0.01) and fewer Proteobacteria (p < 0.05) than did maternal saliva. Many genera previously associated with dental and periodontal infections, e.g. Neisseria, Gemella, Rothia, Actinomyces, Fusobacterium, and Leptotrichia, were more abundant in mothers than in infants. CONCLUSIONS: Salivary microbiota of Tsimane infants and young children up to two years of age do not appear closely related to those of their mothers, despite frequent premastication and preliminary evidence that maternal bacteria is transmitted to premasticated foods. Infant physiology and diet may constrain colonization by maternal bacteria, including several oral pathogens.

Draft Genome Sequence of Thauera sp. Strain SWB20, Isolated from a Singapore Wastewater Treatment Facility Using Gel Microdroplets.

We report here the genome sequence of Thauera sp. strain SWB20, isolated from a Singaporean wastewater treatment facility using gel microdroplets (GMDs) and single-cell genomics (SCG). This approach provided a single clonal microcolony that was sufficient to obtain a 4.9-Mbp genome assembly of an ecologically relevant Thauera species.

Adaptation genomics of a small-colony variant in a Pseudomonas chlororaphis 30-84 biofilm.

The rhizosphere-colonizing bacterium Pseudomonas chlororaphis 30-84 is an effective biological control agent against take-all disease of wheat. In this study, we characterize a small-colony variant (SCV) isolated from a P. chlororaphis 30-84 biofilm. The SCV exhibited pleiotropic phenotypes, including small cell size, slow growth and motility, low levels of phenazine production, and increased biofilm formation and resistance to antimicrobials. To better understand the genetic alterations underlying these phenotypes, RNA and whole-genome sequencing analyses were conducted comparing an SCV to the wild-type strain. Of the genome's 5,971 genes, transcriptomic profiling indicated that 1,098 (18.4%) have undergone substantial reprograming of gene expression in the SCV. Whole-genome sequence analysis revealed multiple alterations in the SCV, including mutations in yfiR (cyclic-di-GMP production), fusA (elongation factor), and cyoE (heme synthesis) and a 70-kb deletion. Genetic analysis revealed that the yfiR locus plays a major role in controlling SCV phenotypes, including colony size, growth, motility, and biofilm formation. Moreover, a point mutation in the fusA gene contributed to kanamycin resistance. Interestingly, the SCV can partially switch back to wild-type morphologies under specific conditions. Our data also support the idea that phenotypic switching in P. chlororaphis is not due to simple genetic reversions but may involve multiple secondary mutations. The emergence of these highly adherent and antibiotic-resistant SCVs within the biofilm might play key roles in P. chlororaphis natural persistence.

Draft Genome Sequence of Enterobacter cloacae Strain S611.

We report draft genomes of Enterobacter cloacae strain S611, an endophytic bacterium isolated from surface-sterilized germinating wheat seeds. We present the assembly and annotation of its genome, which may provide insights into the metabolic pathways involved in adaptation.

Capturing and cultivating single bacterial cells in gel microdroplets to obtain near-complete genomes.

Assembling a complete genome from a single bacterial cell, termed single-cell genomics, is challenging with current technologies. Recovery rates of complete genomes from fragmented assemblies of single-cell templates significantly vary. Although increasing the amount of genomic template material by standard cultivation improves recovery, most bacteria are unfortunately not amenable to traditional cultivation, possibly owing to the lack of unidentified, yet necessary, growth signals and/or specific symbiotic influences. To overcome this limitation, we adopted and modified the method of cocultivation of single-captured bacterial cells in gel microdroplets (GMDs) to improve full genomic sequence recovery. By completing multiple genomes of two novel species derived from single cells, we demonstrated its efficacy on diverse bacterial species using human oral and gut microbiome samples. Here we describe a detailed protocol for capturing single bacterial cells, cocultivating them in medium and isolating microcolonies in GMDs with flow cytometry. Beginning with preliminary studies, obtaining GMDs with single microcolonies for whole-genome amplification may take ∼4 weeks.

Draft Genome Sequence of Pseudomonas putida Strain S610, a Seed-Borne Bacterium of Wheat.

We report the genome sequence of a seed-borne bacterium, Pseudomonas putida strain S610. The size of the draft genome sequence is approximately 4.6 Mb, which is the smallest among all P. putida strains sequenced to date.

Using phage display selected antibodies to dissect microbiomes for complete de novo genome sequencing of low abundance microbes.

BACKGROUND: Single cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. Empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes. METHODS: We describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting (FACS). Single chain (scFv) antibodies are selected using phage display against a bacteria or microbial community, resulting in species-specific antibodies that can be used in FACS for relative quantification of an organism in a community, as well as enrichment or depletion prior to genome sequencing. RESULTS: We selected antibodies against Lactobacillus acidophilus and demonstrate a FACS-based approach for identification and enrichment of the organism from both laboratory-cultured and commercially derived bacterial mixtures. The ability to selectively enrich for L. acidophilus when it is present at a very low abundance (<0.2%) leads to complete (>99.8%) de novo genome coverage whereas the standard single-cell sequencing approach is incomplete (<68%). We show that specific antibodies can be selected against L. acidophilus when the monoculture is used as antigen as well as when a community of 10 closely related species is used demonstrating that in principal antibodies can be generated against individual organisms within microbial communities. CONCLUSIONS: The approach presented here demonstrates that phage-selected antibodies against bacteria enable identification, enrichment of rare species, and depletion of abundant organisms making it tractable to virtually any microbe or microbial community. Combining antibody specificity with FACS provides a new approach for characterizing and manipulating microbial communities prior to genome sequencing.

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome.

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation allows culturing of a diverse microbial community and provides hundreds to thousands of genetically identical cells as input for an MDA reaction. We demonstrate the utility of this approach by comparing sequencing results of gel microdroplets and single cells following MDA. Bias is reduced in the MDA reaction and genome sequencing, and assembly is greatly improved when using gel microdroplets. We acquired multiple near-complete genomes for two bacterial species from human oral and stool microbiome samples. A significant amount of genome diversity, including single nucleotide polymorphisms and genome recombination, is discovered. Gel microdroplets offer a powerful and high-throughput technology for assembling whole genomes from complex samples and for probing the pan-genome of naturally occurring populations.

Artificial polyploidy improves bacterial single cell genome recovery.

BACKGROUND: Single cell genomics (SCG) is a combination of methods whose goal is to decipher the complete genomic sequence from a single cell and has been applied mostly to organisms with smaller genomes, such as bacteria and archaea. Prior single cell studies showed that a significant portion of a genome could be obtained. However, breakages of genomic DNA and amplification bias have made it very challenging to acquire a complete genome with single cells. We investigated an artificial method to induce polyploidy in Bacillus subtilis ATCC 6633 by blocking cell division and have shown that we can significantly improve the performance of genomic sequencing from a single cell. METHODOLOGY/PRINCIPAL FINDINGS: We inhibited the bacterial cytoskeleton protein FtsZ in B.subtilis with an FtsZ-inhibiting compound, PC190723, resulting in larger undivided single cells with multiple copies of its genome. qPCR assays of these larger, sorted cells showed higher DNA content, have less amplification bias, and greater genomic recovery than untreated cells. SIGNIFICANCE: The method presented here shows the potential to obtain a nearly complete genome sequence from a single bacterial cell. With millions of uncultured bacterial species in nature, this method holds tremendous promise to provide insight into the genomic novelty of yet-to-be discovered species, and given the temporary effects of artificial polyploidy coupled with the ability to sort and distinguish differences in cell size and genomic DNA content, may allow recovery of specific organisms in addition to their genomes.